The investigator proposes a novel route to discover bioactive natural compounds. Taking advantage of the fact that of the bacteria present in natural environments, most are not cultivatable in the laboratory, she will make a Bacterial Artificial Chromosome (BAC) library from DNA isolated from soil and possibly from other environments. Current estimates of the number of bacteria per gram of soil are about 10(11). If there are about 10(4) species represented, there will be about 10(7) cells of each kind. This is close to the number of cells in a colony and should provide enough DNA for cloning. The library will be introduced into E. coli, an organism which is relatively promiscuous in the genes it will express; thus there should be relatively few barriers to the expression of genes in this library. The diversity of the library will be tested by examining the phenotypes of the clones in it and by screening by PCR for the unique rDNA signatures of particular phyla of bacteria. The resulting transformants will be screened for the production of compounds with antimicrobial activity. Prof. Handelsman estimates that a library of 500,000 clones is easily obtainable, and if only 0.1% show some activity, the search will have provided a rich yield. The clones will be grown in 48 well microtiter dishes and the culture supernatants in each group will be spotted onto petri plates sown with tester bacteria; the first tests will be carried out against Staphylococcus and Enterococcus, since resistance of these organisms to existing antibiotics has become a significantly clinical problem. Any compounds that cause a ring of growth inhibition will be tested further.